1,5-pentanediamine (also known as 1,5-pentamethylenediamine, abbreviated as diaminopentane) is an important five-carbon compound in the chemical industry, and is mainly used to produce polyamides, polyurethanes and the like. Additionally, it is also used to produce isocyanates, pyridine, piperidine and other important chemical raw materials.
To date, diamines are chemically produced from petroleum-based starting materials via dicarboxylic acid intermediates or by chemical decarboxylation of amino acids (Albrecht, Klaus, et al., Plastics (5th edition), Winnacker-Kuechler, 2005). Due to the limitations of fossil raw materials and increasing attention on green and environmental protection, it becomes ideal to synthesize diamines through bioengineering processes by utilizing renewable raw materials.
There are two main methods for synthesizing pentanediamine by biological processes. One is to obtain pentanediamine by converting glucose through complex metabolic regulation with microbes, that is, to produce pentanediamine from lysine-producing microorganisms by introducing an optional gene encoding lysine decarboxylase, as described in the following reference document (Tabor, Herbert, et al.; Journal of bacteriology, 144 (3), 952-956, 1980). In 2007, Takashi et al. reported that overexpression of lysine decarboxylase by Corynebacterium glutamicum having high ability of lysine synthesis resulted in direct production of pentanediamine by the bacteria during the fermentation with a yield of 2.9 g/l. In studies for improving the yield of pentanediamine, researchers attempted to use Escherichia coli strains with plasmids which overexpress homologous host lysine decarboxylase (cadA gene) (see Japanese Patent Application No. JP2002223770A, JP2008104453A, etc.). In Chinese Patent Application No. CN200810005332, it is reported that a lysine-producing bacteria, Corynebacterium glutamicum, which overexpresses lysine decarboxylase, is used to produce pentanediamine by fermentation with a yield of about 3.4 g/L in the culture broth. In summary, although the process of producing pentanediamine via direct fermentation of glucose with microbes is simple, it has a long fermentation cycle and the bacteria therein have a limited tolerance to intracellular pentanediamine, resulting in a lower yield of pentanediamine which is unfavorable for a large—scale industrial production.
In further studies, researchers developed another method for producing pentanediamine, that is, culturing a strain overexpressing lysine decarboxylase, and collecting the obtained lysine decarboxylase as a catalyst to catalyze lysine into pentanediamine (see JP2002-223771A, JP2004-000114A, EP1482055B1, JP2005-060447A, etc.). Another example is the use of Escherichia coli bacteria which overexpress cadA gene to catalyze lysine adipate and to control the pH during the reaction to improve the stability of the enzyme as described in Chinese Patent Application No. CN101578256A. In Chinese Patent Application No. CN102056889A, it is reported that lysine carbonate is used as a substrate to produce pentanediamine by catalysis, and the carbon dioxide released during the reaction is recycled to control the pH. As described in Chinese Patent No. CN102782146A, a strain expressing cadA gene is subjected to disruption treatment to catalyze exogenous lysine to produce pentanediamine, so as to increase the yield of pentanediamine.
In the above applications or patents, in the process of catalyzing lysine by lysine decarboxylase to produce pentanediamine, a free lysine decarboxylase or cells containing lysine decarboxylase are generally used. Japanese Patent Application No. 2004298033A describes that carrageenan is used to embed lysine decarboxylase-containing strains and then the strains are fermented to produce the enzyme, and then purified lysine salts are catalyzed by the cultured immobilized cells to produce pentanediamine, with about 30% molar conversion of lysine hydrochloride (246 g/L of lysine hydrochloride was catalyzed by the immobilized cells for 150 hours to give pentanediamine at a concentration of 40 g/l).
It is reported that 3 wt % ca-alginate is used to immobilize cells containing lysine decarboxylase, resulting in a poor stability for the immobilized cell. In particular, the enzyme activity in the second time decreased significantly, and in the forth time, the activity of the enzyme decreased to about 38% of the first time (Jiang Lili et al., “Producing Cadaverine by Cell immobilization of Lysine Decarboxylase”, Fine Chemicals, 2007, 24 (11), 1080-1084).
In the actual processes, natural polymer gels such as sodium alginate and the like present problems such as a lower strength, being easy to be decomposed by microorganisms, being deformed, disrupted or dissolved during the transformation process, causing leakage of enzymes or cells, a low re-use efficiency of immobilized enzyme, and others.
Cells having lysine decarboxylase activity can catalyze various forms of lysine solutions, for example, purified lysine, hydrochloride, sulfate, phosphate, adipate and other salts of lysine, and unpurified lysine fermentation broth. However, due to the complex composition of lysine fermentation broth, stability of the cells containing lysine decarboxylase is greatly impacted, easily leading to cell lysis, enzyme loss, and reducing utilization efficiency of lysine decarboxylase. Immobilization of the cells containing lysine decarboxylase in a certain carrier will significantly improve the stability of the cells containing lysine decarboxylase in the lysine fermentation broth, improve the catalytic efficiency of the enzyme, and reduce lysine purification costs.
In summary, there are a number of studies on the production of pentanediamine by catalyzing lysine using lysine decarboxylase produced by microorganisms, which are characterized by most use of purified lysine or lysine salt, the need for lysine purification, complex process, and high production costs, and thus limiting the industrial production of pentanediamine. In addition, the reaction system for production of pentanediamine by enzymatic catalysis of lysine mostly uses free enzyme-containing cells, which have low enzyme stability, leading to complex extraction process for pentanediamine in the enzyme reaction solution, affecting the purity of the finished product of pentanediamine, and being not conducive to industrial production.
Accordingly, there is a need in the art for a process that is simpler and more economical and can improve the efficiency of re-use of lysine decarboxylase.